U.S. patent application number 12/024403 was filed with the patent office on 2008-08-07 for photocrosslinked-polysaccharide composition and production process of the same.
This patent application is currently assigned to Seikagaku Corporation. Invention is credited to Tomoya SATO.
Application Number | 20080188585 12/024403 |
Document ID | / |
Family ID | 32984507 |
Filed Date | 2008-08-07 |
United States Patent
Application |
20080188585 |
Kind Code |
A1 |
SATO; Tomoya |
August 7, 2008 |
PHOTOCROSSLINKED-POLYSACCHARIDE COMPOSITION AND PRODUCTION PROCESS
OF THE SAME
Abstract
A process for producing a photocrosslinked-polysaccharide
composition, which comprises: freezing a photoreactive
polysaccharide-containing solution comprising a photoreactive
polysaccharide in which a photoreactive group is bound to a
polysaccharide, an aqueous solvent capable of dissolving the
photoreactive polysaccharide, and any one substance selected from
the group consisting of alcohol having compatibility with the
aqueous solvent, a surfactant and a chelating agent; and
irradiating the resulting frozen product with light, and a
photocrosslinked-polysaccharide composition obtained by the
process.
Inventors: |
SATO; Tomoya;
(Higashiyamato-shi, JP) |
Correspondence
Address: |
SUGHRUE-265550
2100 PENNSYLVANIA AVE. NW
WASHINGTON
DC
20037-3213
US
|
Assignee: |
Seikagaku Corporation
Chiyoda-ku
JP
|
Family ID: |
32984507 |
Appl. No.: |
12/024403 |
Filed: |
February 1, 2008 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
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10548533 |
Feb 28, 2006 |
7365059 |
|
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PCT/JP2004/003204 |
Mar 11, 2004 |
|
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12024403 |
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Current U.S.
Class: |
522/84 |
Current CPC
Class: |
C08J 2305/00 20130101;
C08K 5/0025 20130101; C08J 3/28 20130101; C08K 5/0025 20130101;
C08L 1/286 20130101 |
Class at
Publication: |
522/84 |
International
Class: |
C08F 290/14 20060101
C08F290/14 |
Foreign Application Data
Date |
Code |
Application Number |
Mar 11, 2003 |
JP |
2003-65704 |
Claims
1-18. (canceled)
19. A photocrosslinked-polysaccharide composition, which comprises:
a photocrosslinked-polysaccharide in an amount of from 0.5 to 10%
(w/w); and at least one substance selected from the group
consisting of an alcohol, a surfactant and a chelating agent having
compatibility with the aqueous solvent in an amount of from 0.5 to
10% (w/w), wherein the photocrosslinked-polysaccharide is a
photocrosslinked-polysaccharide in which a polysaccharide bound to
a photoreactive group is crosslinked by light irradiation in the
presence of the substance having compatibility with the aqueous
solvent, wherein the alcohol is represented by the following
formula (I): R--OH (I) wherein R in the formula is selected from
the group consisting of: straight chain alkyl having from 1 to 10
carbon atoms; branched chain alkyl having from 3 to 10 carbon
atoms; ##STR00003## wherein the surfactant is selected from the
group consisting of poly(ethylene glycol) and alkyl sulfate, and
wherein the chelating agent is selected from the group consisting
of hydroxycarboxylic acid and polyaminocarboxylic acid.
20. The photocrosslinked-polysaccharide composition according to
claim 19, wherein the polysaccharide has a weight average molecular
weight of from 1,500 to 500,000.
21. The photocrosslinked-polysaccharide composition according to
claim 19, wherein the alcohol is represented by the following
formula (I): R--OH (I) wherein R in the formula (I) is selected
from the group consisting of: ##STR00004##
22. The photocrosslinked-polysaccharide composition according to
claim 19, wherein the photoreactive group comprises a derivative in
which aminopropanol and cinnamic acid are bound, represented by the
following formula: H.sub.2N(CH.sub.2).sub.3OCOCH.dbd.CH-Ph wherein
Ph represents a phenyl group; and wherein the polysaccharide is
selected from the group consisting of chondroitin sulfate, dermatan
sulfate, heparin, 6-O-desulfated heparin and
carboxymethylcellulose.
23. The photocrosslinked-polysaccharide composition according to
claim 19, wherein the alcohol, the surfactant or the chelating
agent is selected from the group consisting of d-sorbitol,
poly(ethylene glycol), sodium lauryl sulfate,
ethylenediaminetetraacetic acid, and citric acid.
24. The photocrosslinked-polysaccharide composition according to
claim 19, which has a viscosity of from 300 to 30,000 mPaS when
measured by a rotational viscometer under conditions of standard
cone (1 degree) and 20.degree. C., and is gel state.
Description
CROSS REFERENCE TO RELATED APPLICATIONS
[0001] This is a continuation of application Ser. No. 10/548,533
filed Feb. 28, 2006, which is a National Stage Entry of
PCT/JP2004/003204 filed on Mar. 11, 2004. The entire disclosure of
the prior application, application Ser. No. 10/548,533, is
considered part of the disclosure of the accompanying continuation
application and is hereby incorporated by reference.
TECHNICAL FIELD
[0002] The present invention relates to a
photocrosslinked-polysaccharide composition in which a
photoreactive polysaccharide to which a photoreactive group is
bound is crosslinked, and a production process of the same.
BACKGROUND ART
[0003] As an example of the technique for obtaining a crosslinked
polysaccharide gel, Eur. J. Pharm. Sci., 2002 March; 15(2): 139-48
describes an example in which chondroitin sulfate (hereinafter also
referred to as "CS") is crosslinked using diglycidyl ether as the
crosslinking agent to form the gel. However, when such a
crosslinking agent is used, crosslinking reaction occurs
simultaneously with the reaction of chondroitin sulfate with the
crosslinking agent, thus causing a problem of having difficulty in
removing the unreacted crosslinking agent from the gel.
[0004] Accordingly, a technique has been developed for
photo-crosslinking a photoreactive polysaccharide from which the
crosslinking agent can be easily removed. For example, a technique
is known in which a photoreactive glycosaminoglycan is obtained by
binding a photoreactive crosslinking group in advance to
glycosaminoglycan, the unreacted crosslinking agent is removed by
purifying the photoreactive glycosaminoglycan, and then
crosslinked-glycosaminoglycan is obtained by irradiating light
(JP-A-6-73102). This literature describes a technique in which
photoreactive chondroitin sulfate is obtained by binding a
photoreactive crosslinking group to chondroitin sulfate, and the
photoreactive chondroitin sulfate is dissolved in phosphate
buffered saline and then crosslinked by irradiating ultraviolet ray
under the state of solution using a mercury lamp to form the gel.
However, when a gel of the crosslinked glycosaminoglycan is
obtained by such a method, since crosslinking efficiency is poor by
the crosslinking reaction under the state of the solution, it is
necessary to irradiate light by preparing an aqueous solution
containing a photoreactive glycosaminoglycan at a high
concentration (10% or more), so that it cannot always be said that
this is a method having good efficiency.
[0005] On the other hand, as a technique for efficiently
crosslinking a photoreactive polysaccharide, for example, a
technique is known in which photoreactive glycosaminoglycan
prepared by binding a photoreactive crosslinking group to
glycosaminoglycan is dissolved in an aqueous solvent, the resulting
solution is frozen, and its crosslinking is carried out by
irradiating light while keeping the frozen state (WO02/060971). By
this method, a crosslinked glycosaminoglycan showing a spongy
(porous) property having markedly superior water taking/discharging
property is specifically obtained, and a crosslinked
glycosaminoglycan showing a gel property is not obtained.
DISCLOSURE OF THE INVENTION
[0006] Concern has been directed toward a process for producing a
photocrosslinked-polysaccharide, by which a photoreactive
polysaccharide prepared by binding a photoreactive group thereto
can be crosslinked efficiently and easily, and gel property can be
obtained.
[0007] In order to solve the above-described problems, the present
inventors have conducted intensive studies and found as a result
that a product having gel property can be easily obtained when a
solution prepared by dissolving a photoreactive polysaccharide and
either one of an alcohol, a surfactant or a chelating agent having
compatibility with an aqueous solvent in the aqueous solvent is
frozen, light is irradiated to the thus obtained frozen product,
and then the irradiated product is melted, if necessary, and that a
photoreactive polysaccharide is crosslinked easily and markedly
efficiently by the process, and the present invention has been
accomplished based on this knowledge.
[0008] That is, the present invention relates to the following (1)
to (18).
(1) A process for producing a photocrosslinked-polysaccharide
composition, which comprises:
[0009] freezing a photoreactive polysaccharide-containing solution
comprising a photoreactive polysaccharide in which a photoreactive
group is bound to a polysaccharide, an aqueous solvent capable of
dissolving the photoreactive polysaccharide, and any one substance
selected from the group consisting of an alcohol, a surfactant and
a chelating agent having compatibility with the aqueous solvent;
and
[0010] irradiating the resulting frozen product with light.
(2) The process according to (1), wherein the alcohol has such a
property that it keeps a frozen state at -7.degree. C. or less when
the alcohol is added to an aqueous solvent solution of the
photoreactive polysaccharide and is frozen to obtain the frozen
product and then the temperature is increased. (3) The process
according to (1) or (2), wherein the alcohol is represented by the
following formula (I):
R--OH (I)
[0011] wherein R in the formula is selected from the group
consisting of:
[0012] straight chain alkyl having from 1 to 10 carbon atoms,
[0013] branched chain alkyl having from 3 to 10 carbon atoms,
##STR00001##
(4) The process according to (1), wherein the surfactant is a
nonionic surfactant or an anionic surfactant. (5) The process
according to (4), wherein the nonionic surfactant is poly(ethylene
glycol), and the anionic surfactant is alkyl sulfate. (6) The
process according to (1), wherein the chelating agent is selected
from the group consisting of hydroxycarboxylic acid and
polyaminocarboxylic acid. (7) The process according to (6), wherein
the hydroxycarboxylic acid is citric acid, and the
polyaminocarboxylic acid is ethylenediaminetetraacetic acid. (8)
The method according to any one of (1) to (7), wherein the
polysaccharide constituting the photoreactive polysaccharide is
selected from the group consisting of heteroglycan, homoglycan and
derivatives thereof. (9) The process according to (8), wherein the
heteroglycan is glycosaminoglycan selected from the group
consisting of hyaluronic acid, chondroitin, chondroitin sulfate,
dermatan sulfate, heparin, heparan sulfate and keratan sulfate, and
the homoglycan is acidic homoglycan selected from the group
consisting of alginic acid and carboxymethylcellulose. (10) The
process according to (8), wherein the glycosaminoglycan derivative
is a desulfation and/or oxidation reduction derivative. (11) The
process according to (10), wherein the desulfation derivative is
6-O-desulfated heparin in which a sulfate group bound to the
6-position hydroxyl group of the N-acetylglucosamine residue of
heparin is removed. (12) The process according to any one of (1) to
(11), wherein the polysaccharide has a weight average molecular
weight of from 1,500 to 500,000. (13) The process according to any
one of (1) to (12), wherein the photoreactive group in the
photoreactive polysaccharide binds to a carboxyl group of the
polysaccharide. (14) The process according to any one of (1) to
(13), wherein the photoreactive polysaccharide-containing solution
is a solution containing from 0.5 to 10% of the photoreactive
polysaccharide. (15) The process according to (14), wherein the
photoreactive polysaccharide is glycosaminoglycan having a
molecular weight of from 1,500 to 100,000. (16) A
photocrosslinked-photocrosslinked-polysaccharide composition which
is obtained by freezing a photoreactive polysaccharide-containing
solution comprising a photoreactive polysaccharide in which a
photoreactive group is bound to a polysaccharide, an aqueous
solvent capable of dissolving the photoreactive polysaccharide and
any one substance selected from the group consisting of an alcohol,
a surfactant and a chelating agent having compatibility with the
aqueous solvent; irradiating the resulting frozen product with
light; and melting the frozen product. (17) The
photocrosslinked-polysaccharide composition according to (16),
which has a viscosity of from 300 to 30,000 mPaS when measured by a
rotational viscometer under conditions of standard cone (1 degree)
and 20.degree. C., and is gel state. (18) A kit for injection of
photocrosslinked-polysaccharide composition, which comprises the
photocrosslinked-polysaccharide composition according to (17) which
is filled in an injector capable of pushing out the
composition.
BRIEF DESCRIPTION OF THE DRAWINGS
[0014] FIG. 1 is a photograph showing properties of
photocrosslinked-CS compositions obtained by adding each of PEG 400
(2%), PEG 4000 (2%), citric acid (2%), citric acid (4%), SDS (4%),
EDTA (4%) and d-sorbitol (8%), and a control
photocrosslinked-CS.
[0015] FIG. 2 is a result of oscillation tests for the
photocrosslinked-polysaccharide compositions obtained by adding
each of PEG 400 (2%), PEG 4000 (2%) and citric acid and for a
control, carried out in Example 6. In the drawing, diamonds
indicate the storage modulus of elasticity (G') of 1 ml sample,
squares indicate the loss modulus of elasticity (G'') of 1 ml
sample, and triangles indicate the loss tangent (tan .delta.:
(G''/G')). The scale of the left side vertical line shows the
stress (Pa; Pascal), and the scale of right side vertical line
shows the loss tangent. The scale of horizontal line shows the
frequency (Hz; Hertz).
[0016] FIG. 3 is a photograph showing properties of
photocrosslinked-CS compositions obtained by adding each of PEG
4000 (2%), SDS (4%) and EDTA (4%), and a control
photocrosslinked-CS.
BEST MODE FOR CARRYING OUT THE INVENTION
[0017] The present invention is described below in detail based on
the embodiments of the present invention.
[0018] The present invention relates to a process for producing a
photocrosslinked-polysaccharide composition, which comprises:
freezing a photoreactive polysaccharide-containing solution
comprising a photoreactive polysaccharide in which a photoreactive
group is bound to a polysaccharide, an aqueous solvent capable of
dissolving the photoreactive polysaccharide, and any one substance
selected from the group consisting of an alcohol, a surfactant and
a chelating agent having compatibility with the aqueous solvent;
and irradiating the resulting frozen product with light
(hereinafter also referred to "production process of the present
invention").
[0019] The present invention further relates to a
photocrosslinked-polysaccharide composition which is obtained by
freezing a photoreactive polysaccharide-containing solution
comprising a photoreactive polysaccharide to which a photoreactive
group is bound, an aqueous solvent capable of dissolving the
photoreactive polysaccharide and any one substance selected from
the group consisting of an alcohol, a surfactant and a chelating
agent having compatibility with the aqueous solvent; irradiating
the resulting frozen product with light; and melting the frozen
product (hereinafter also referred to as "composition of the
present invention").
[0020] The polysaccharide which constitutes the photoreactive
polysaccharide to be used in the production process of the present
invention includes heteroglycan, homoglycan and derivatives
thereof. Also, the polysaccharide which constitutes the
photoreactive polysaccharide is the same as the polysaccharide
which constitutes the photocrosslinked-polysaccharide in the
composition of the present invention.
[0021] The heteroglycan is a polysaccharide comprising two or more
different monosaccharides as the constituting saccharides, and
according to the present invention, and glycosaminoglycan or
derivatives thereof are particularly preferable. The
glycosaminoglycan (hereinafter also referred to as "GAG") is a
polysaccharide comprising a basal structure containing an amino
sugar and uronic acid (or galactose). Examples include hyaluronic
acid, chondroitin, chondroitin sulfate, dermatan sulfate, keratan
sulfate, heparin and heparan sulfate; preferred examples include
chondroitin, chondroitin sulfate, dermatan sulfate and heparin; and
more preferred examples include chondroitin sulfate, dermatan
sulfate and heparin.
[0022] Examples of the derivatives of the glycosaminoglycans
include sulfation derivatives in which a sulfate group is bound to
GAG, desulfation derivatives in which sulfate group is partially or
completely removed from GAG, oxidation reduction derivatives in
which GAG is subjected to oxidation reduction reaction, oxidation
reduction-desulfation derivatives in which GAG is subjected to
oxidation reduction reaction and desulfation reaction, and the
like. The sulfation derivatives include sulfated hyaluronic acid,
chondroitin polysulfate and the like. The desulfaction derivatives
include 6-position desulfated heparin (WO00/06608), 2-position
desulfated heparin (JP-A-2003-113090), completely desulfated
heparin and the like. The oxidation reduction-desulfation
derivatives include periodic acid oxidation reduction-desulfation
heparin (JP-A-11-310602) and the like. Among these, desulfation
derivatives and oxidation reduction-desulfation derivatives are
preferable, and desulfation derivatives are particularly
preferable.
[0023] On the other hand, the homoglycan is a polysaccharide
comprising a single monosaccharide as the constituting saccharide.
Examples in the present invention include glucan (amylose,
cellulose, etc.), mannan, glycuronan (pectic acid, alginic acid,
etc.), polyglycosamine (chitin, colominic acid, etc.),
polygalactosamine and the like; preferred examples include glucan,
alginic acid and the like; and more preferred examples include
cellulose and alginic acid.
[0024] Examples of the derivatives of the homoglycan include
carboxymethyl derivatives such as carboxymethylcellulose;
hydroxymethyl derivatives such as hydroxymethylcellulose;
deacetylation derivatives such as chitosan; and the like. According
to the present invention, derivatives having water-solubility are
particularly preferable, so that carboxymethyl derivatives and
hydroxymethyl derivatives are preferable; carboxymethyl derivatives
are more preferable; and carboxymethylcellulose (hereinafter also
referred to as "CMC") is most preferable.
[0025] Also, each of alginic acid and CMC is acidic homoglycan.
[0026] In addition, two or more of the polysaccharides exemplified
in the above can be used as a mixture in the present invention.
[0027] The polysaccharide to be used in the present invention has a
weight average molecular weight of preferably from 1,500 to
500,000, more preferably from 1,500 to 250,000, further preferably
from 1,500 to 150,000, and most preferably from 1,500 to
100,000.
[0028] Also, the technique for obtaining gel-state
photocrosslinked-GAG by irradiating with light a solution prepared
by dissolving photoreactive GAG prepared by binding a photoreactive
group to GAG having a relatively low molecular weight for
crosslinking was previously difficult to carry out because of the
difficulty in setting conditions and the like. However, when the
production process of the present invention is used, a gel-state
photocrosslinked-polysaccharide composition can be obtained
markedly easily even when a photoreactive polysaccharide comprising
a polysaccharide having a relatively low molecular weight is
used.
[0029] The polysaccharide to be used in the present invention may
be derived from a natural origin or a product chemically
synthesized or produced by a microorganism such as yeast by genetic
engineering techniques. Regarding GAG, it is possible and
preferable in general to prepare it by extracting from a biotic
material (cockscomb, umbilical cord, cartilage, skin, small
intestine, blood vessel or the like).
[0030] The photoreactive group (photoreactive crosslinking group)
which constitutes the photocrosslinked-polysaccharide of the
present invention is a crosslinking group comprising a
photoreactive residue which constitutes the photoreactive
polysaccharide. The photoreactive residue may be a residue of any
compound which generates a photo-dimerization reaction or a
photo-polymerization reaction by ultraviolet ray irradiation, and
specific examples include cinnamic acid, substituted cinnamic acid,
acrylic acid, acrylic acid derivatives, maleic acid, fumaric acid,
sorbic acid, coumarin, thymine and the like. Also, the substituted
cinnamic acid includes aminocinnamic acid (preferably
p-aminocinnamic acid), which is cinnamic acid in which any one of
hydrogen atoms of the benzene ring is substituted with an amino
group, and the acrylic acid derivatives include thiopheneacrylic
acid, furylacrylic acid and the like.
[0031] Among these photoreactive residues, a substance having a
vinylene group which can form a cyclobutane ring by photoreaction
is preferable, and from the viewpoint of photo-reactivity and
safety, cinnamic acid or substituted cinnamic acid is particularly
preferable, and aminocinnamic acid is particularly preferable as
the substituted cinnamic acid.
[0032] In addition, according to the present invention, a spacer
may be contained for keeping a predetermined distance by binding to
both of the photoreactive residue and polysaccharide when the
photoreactive polysaccharide is formed. Regarding the photoreactive
group (photoreactive crosslinking group) to be used in the present
invention, the photoreactive residue and a spacer may be bound, and
it is preferable that it is bound to the polysaccharide via a
spacer. Accordingly, derivatives in which a spacer is bound to
cinnamic acid or a substituted cinnamic acid are most preferable as
the photoreactive group and are also most preferable as the
photoreactive crosslinking group.
[0033] According to the present invention, a functional group which
generates photo-dimerization reaction or photo-polymerization
reaction by photo-reaction of cinnamic acid, substituted cinnamic
acid, thymine or the like is defined as "photoreactive residue",
and both cases of the photoreactive residue alone and a substituent
in which a spacer is bound to the photoreactive residue are defined
as "photoreactive group" or "photoreactive crosslinking group".
[0034] Specific examples of the most preferable photoreactive
crosslinking group as described in the above include cinnamic acid
aminoalkyl ester derivatives in which aminoalcohol
(H.sub.2N--(CH.sub.2).sub.n--OH; n=from 1 to 18, or
H.sub.2N--(CH.sub.2--O).sub.m--CH.sub.2--OH; m=from 1 to 9) is
bound to the carboxyl group of cinnamic acid via an ester bond,
derivatives in which diamine (H.sub.2N--(CH.sub.2).sub.1--NH.sub.2;
1=from 1 to 10) or diol (HO--(CH.sub.2).sub.k--OH; k=from 1 to 10)
is introduced into cinnamic acid or substituted cinnamic acid,
derivatives in which amino acid (HOOC--(CHR).sub.j--NH.sub.2;
j=from 1 to 10), a peptide or the like is introduced into
substituted cinnamic acid (aminocinnamic acid), and the like. Among
these, derivatives in which an amino-alcohol is introduced into the
carboxyl group of cinnamic acid (cinnamic acid aminoalkyl ester)
are preferable, and regarding the amino-alcohol, n in the
above-described formula is preferably from 1 to 18, particularly
preferably from 3 to 6, and extremely preferably 3 or 4.
[0035] The region where the polysaccharide and photoreactive group
are bound in the photoreactive polysaccharide to be used in the
production process of the present invention, namely the region
where the polysaccharide of the photocrosslinked-polysaccharide and
the photoreactive group in the composition of the present invention
are bound, can be selected based on the functional groups
respectively possessed by the polysaccharide to be used and the
photoreactive group (photoreactive crosslinking group) to be used,
and are not particularly limited. For example, when GAG, such as
hyaluronic acid, chondroitin, chondroitin sulfate, dermatan
sulfate, heparin, heparan sulfate or a derivative thereof, is used
as the polysaccharide, the binding region includes an amino group
(exists as an acetylamino group or a sulfamino group), a hydroxyl
group, a carboxyl group and the like, and a carboxyl group is
particularly preferable. As an example, when cinnamic acid
aminoalkyl ester is used as the photoreactive group (photoreactive
crosslinking group), and GAG or a derivative thereof is used as the
polysaccharide, the photoreactive group (photoreactive crosslinking
group) is bound to the GAG via an amide bond of the amino group of
aminoalkyl with the carboxyl group of the GAG or a derivative
thereof.
[0036] The introduction ratio (hereinafter referred to as
substitution degree) of the photoreactive group of the
photoreactive polysaccharide to be used in the production process
of the present invention is from 0.1 to 75%, preferably from 0.3 to
70%, and as described above, the substitution degree of the
photoreactive group of the photoreactive polysaccharide according
to the composition of the present invention is also from 0.1 to
75%, preferably from 0.3 to 70%. The introduction reaction of the
photoreactive group (photoreactive crosslinking group) into
polysaccharide can be carried out, for example, in accordance with
the method described in JP-A-6-073102 or the like, and the value of
this substitution degree can be optionally changed or adjusted by
controlling the mol number of the polysaccharide and the mol number
of the photoreactive group (photoreactive crosslinking group), to
be used in the reaction. Also, the substitution degree can be
measured by the method described in Measurement Example 1 which is
described later.
[0037] Any one of the substances selected from the group consisting
of alcohol, a surfactant and a chelating agent according to the
present invention (hereinafter also referred to as "additive
substance of the present invention") is not particularly limited,
so long as it has compatibility with an aqueous solvent.
[0038] The alcohol includes the alcohol represented by the
following formula (I):
R--OH (I)
[0039] wherein R in the above formula is selected from the group
consisting of:
[0040] straight chain alkyl having from 1 to 10 carbon atoms,
[0041] branched chain alkyl having from 3 to 10 carbon atoms,
##STR00002##
[0042] The straight chain alkyl having from 1 to 10 carbon atoms
includes methyl, ethyl and the like, and the branched chain alkyl
having from 3 to 10 carbon atoms includes isopropyl, t-butyl and
the like.
[0043] The alcohol includes lower alcohol, polyhydric alcohol or
sugar alcohol.
[0044] The lower alcohol includes alcohol having from 1 to 10, more
preferably from 1 to 8, carbon atoms, and specific examples include
methanol, ethanol, isopropyl alcohol, t-butyl alcohol and the like.
Examples of the polyhydric alcohol include alcohol in which two or
more hydroxyl groups are present in the molecule, more preferably
alcohol having three or more hydroxyl groups. Examples include
ethylene glycol and glycerol, and ethylene glycol (PEG) is
preferable. In addition, it is possible to use either chain sugar
alcohol or cyclic sugar alcohol as the sugar alcohol, but chain
sugar alcohol is preferable. The sugar alcohol includes inositol,
mannitol, xylitol, sorbitol and the like, preferred examples
include mannitol, xylitol and sorbitol, and more preferred examples
include mannitol and sorbitol.
[0045] The surfactant is preferably a nonionic surfactant or an
anionic surfactant, the nonionic surfactant is more preferably
polyethylene glycol, and the anionic surfactant is more preferably
alkyl sulfate, most preferably sodium dodecyl sulfate.
[0046] The chelating agent includes hydroxycarboxylic acid such as
citric acid, and polyaminocarboxylic acid such as
ethylenediaminetetraacetic acid.
[0047] In addition, it is preferable that the additive agent of the
present invention, particularly the above-described alcohol, has
such a property that when the additive agent of the present
invention is added to an aqueous solvent solution of the
photoreactive polysaccharide and frozen to obtain a frozen product
and then the temperature is increased, the frozen product keeps the
frozen state at -7.degree. C. or less. In this case, the frozen
state means a completely coagulated state. In addition, in order to
produce the composition of the present invention having gel
property, the additive agent of the present invention is suitably
used because of its performance in which the
photocrosslinked-product formed when light irradiation is carried
out in accordance with the production process of the present
invention does not become an insoluble solid matter.
[0048] Also, as is described later, it is possible to use the
composition of the present invention in medical treatment or
medicinal use, and when used for such a purpose, it is necessary to
select a substance having at least aqueous solvent compatibility
and high biocompatibility as the additive agent of the present
invention.
[0049] The solvent of the photoreactive polysaccharide-containing
solution in the production process of the present invention is an
aqueous solvent which can dissolve the photoreactive polysaccharide
and is not particularly limited with the proviso that it contains
water, and the solution may contain a salt. That is, as water or an
aqueous solvent comprising a solution containing water, a solvent
capable of dissolving the photoreactive polysaccharide and the
additive agent of the present invention can be used.
[0050] As the aqueous solvent, water, water for injection or
physiological saline, or a buffer such as Tris-HCl buffer or
phosphate buffered saline can be used.
[0051] The concentration of the photoreactive polysaccharide in the
photoreactive polysaccharide-containing solution according to the
production process of the present invention is, for example, from
0.5 to 10% (w/w), preferably from 0.8 to 8% (w/w), and most
preferably from 1 to 6% (w/w). Also, the concentration of the
additive agent of the present invention in the photoreactive
polysaccharide-containing solution is, for example, from 0.5 to 10%
(w/w), more preferably from 0.8 to 9% (w/w), and most preferably
from 1 to 8% (w/w).
[0052] Also, as a preferred embodiment of the present invention,
when considered based on the relationship between the molecular
weight of the polysaccharide constituting the photoreactive
polysaccharide and the concentration of the photoreactive
polysaccharide in the solution, it is preferable that the molecular
weight is from 1,500 to 100,000 and the concentration is 0.5 to
10%, it is further preferable that the molecular weight is 1,500 to
100,000 and the concentration is 0.8 to 8%, and it is most
preferable that the molecular weight is 1,500 to 100,000 and the
concentration is 1 to 6%. Under the conditions as described in the
above, it is difficult to obtain a gel-state
photocrosslinked-polysaccharide by the conventional
photo-crosslinking methods, but a gel-state composition can be
easily obtained by the production process of the present
invention.
[0053] The temperature conditions for carrying out freezing of the
photoreactive polysaccharide-containing solution are not
particularly limited. The freezing does not require complete
coagulation of the solution, and it may be under such temperature
conditions that it forms a so-called "sherbet state" in which the
solution is partially coagulated to form a solid-liquid mixed
phase. Thus, the "freezing" according to the production process of
the present invention is a general idea which includes such a
solid-liquid mixed phase state. However, the most preferable
"temperature conditions for carrying out freezing" are temperature
conditions under which the solution is completely coagulated and
solidified, and are, for example, 0.degree. C. or less, preferably
-5.degree. C. or less, more preferably -7.degree. C. or less,
further preferably -15.degree. C. or less, most preferably
-20.degree. C. or less.
[0054] It is preferable that the irradiation of light is carried
out under such conditions that the photoreactive residue
efficiently generates photo-dimerization reaction or
photo-polymerization reaction. Regarding the kind of light to be
irradiated, ultraviolet ray can be preferably exemplified, and as
the ultraviolet ray, ultraviolet ray having a wavelength (e.g.,
from 200 to 600 nm) which does not cut glycoside bonds of the
polysaccharide and generates photo-crosslinking reaction on the
photoreactive residue is selected. As the ultraviolet lamp, a high
pressure mercury lamp or a metal halide lamp is preferable. In
addition, it is preferable that unnecessary wavelengths of the
ultraviolet rays generated from such a lamp are eliminated, for
example, using a cut-off filter or the like. As the cut-off filter,
a process filter for exclusive use is preferable, but a
commercially available hard glass can also sufficiently cope with
the purpose when it can cut off the unnecessary wavelengths.
[0055] It is necessary that the quantity of irradiation light is a
quantity of light by which the photoreactive polysaccharide can
sufficiently generate photo-crosslinking reaction, which is
specifically 500 mJ/cm.sup.2 or more, preferably 2,500 mJ/cm.sup.2
or more, and most preferably 4,000 mJ/cm.sup.2 or more, by an
ultraviolet ray of 280 nm. Also, when an actinometer is used by
attaching a 1/10 extinction filter, the numerical value on the
actinometer is displayed by a value of 1/10 of the actual quantity
of irradiation light, and the above-described range is the actual
quantity of irradiation light.
[0056] The composition of the present invention is a
photocrosslinked-polysaccharide composition which is obtained by
the production process of the present invention, and, as described
above, it can be obtained by freezing a solution which contains at
least a photoreactive polysaccharide prepared by binding a
photoreactive group thereto, an aqueous solvent capable of
dissolving the photoreactive polysaccharide and the additive agent
of the present invention, irradiating the resulting frozen product
with light, and then melting the frozen product.
[0057] That is, the composition of the present invention is a
photocrosslinked-polysaccharide composition which comprises a
photocrosslinked-polysaccharide, an aqueous solvent capable of
dissolving the photoreactive polysaccharide and the additive agent
of the present invention having aqueous solvent compatibility, and
since the composition of the present invention is produced by
freezing the aqueous solvent-containing solution, carrying out the
photo-crosslinking reaction and then melting the same, it contains
water.
[0058] The composition of the present invention has high
viscoelasticity in comparison with the photoreactive
polysaccharide-containing solution and shows a gel property as a
hydrogel abundantly containing water or a spongy property excellent
in water taking/discharging property, and one showing gel property
is preferable. In general, it is difficult to clearly classify
properties of substances having viscoelasticity using numerical
values such as gel property or spongy property, but physical
properties of gel based on the viscoelasticity can be shown by
using the dynamic moduli of elasticity such as storage modulus of
elasticity (G'), loss modulus of elasticity (G'') or loss tangent
(tan .delta.: (G''/G'), and the viscosity.
[0059] For example, when the composition of the present invention
is used by filling it in an injector such as a syringe for
injection, as is described below, the composition of the present
invention having gel property is suitable, particularly one having
the following moduli of elasticity or viscosity is preferable. When
the moduli of elasticity measured by control stress type rheometer
(manufactured by Carri-Med, England) are used as the indexes, and
when measured by continuously changing from 0.01 to 10 Hz at
37.degree. C., the storage modulus of elasticity (G') is preferably
from 0.1 to 800 Pa, the loss modulus of elasticity (G'') is
preferably from 1 to 180 Pa, and the loss tangent (G''/G') is
preferably from 0.1 to 0.8. Also, it is known that when the storage
modulus of elasticity is high and the loss modulus of elasticity is
low, it becomes a hard gel having the strong elasticity, and in the
opposite case, it becomes a gel having strong viscosity. In
addition, when the viscosity of 1 ml of the composition of the
present invention measured by a rotational viscometer under
conditions of standard cone (1 degree) and 20.degree. C. is used as
the index, it is preferably from 300 to 30,000 mPaS, and more
preferably from 300 to 20,000 mPaS.
[0060] The photocrosslinked-polysaccharide of the composition of
the present invention shows a crosslinking ratio of from 0.5 to
80%, more preferably from 1.5 to 75%, and most preferably from 5 to
70%. Also, the crosslinking ratio is expressed as a ratio
(percentage) of 2 times of the number of moles of the dimer to the
number of moles of photoreactive crosslinking group introduced into
the polysaccharide. For example, when cinnamic acid or substituted
cinnamic acid is used as the photoreactive residue and GAG is used
as the polysaccharide, the crosslinking ratio can be calculated by
the method described in Measurement Example 2.
[0061] The concentration of the photocrosslinked-polysaccharide in
the composition of the present invention is the same as the
concentration of the photoreactive polysaccharide in the
above-described photoreactive polysaccharide-containing solution,
and is, for example, from 0.5 to 10% (w/w), preferably from 0.8 to
8% (w/w), and most preferably from 1 to 6% (w/w). Also,
concentration of the additive agent of the present invention in the
composition of the present invention is almost the same as the
concentration of the additive agent of the present invention in the
photoreactive polysaccharide-containing solution, and is, for
example, from 0.5 to 10% (w/w), more preferably from 0.8 to 9%
(w/w), and most preferably from 1 to 8% (w/w).
[0062] It is possible to use the composition of the present
invention, for example, in a medical material for protecting a
wound area (a wound covering material), a medical material for
keeping a space in the living body (a space keeping material), a
medical material for filling a cavity of connective tissue such as
bone (a bone filling material), an artificial body fluid (an
artificial joint fluid, an artificial tear fluid, an operation
assisting material for opthalmology or the like), a material for
keeping moisture on the living body surface or the like (moisture
keeping material), an additive agent which is added for keeping the
dosage form of a medicament or the like (a filler), a base material
to be used as a foothold of cells in cell culture (base material
for cell culture) and the like. Particularly, the composition of
the present invention having gel property is more suitable to be
used in the above-described applications due to its characteristic
property of being a fluid state having high viscosity, and it is
also possible to use the composition of the present invention which
is filled in an injector capable of pushing out the composition, as
a kit for injection which is used in the above-described medical
applications.
[0063] Also, the injector capable of pushing out the composition is
an injector which is equipped with a plunger or the like for drug
extrusion and can push out the filled drug, and it is possible to
use a generally used tool.
[0064] The present invention is described below in more detail.
MEASUREMENT EXAMPLE 1
Method for Measuring Substitution Degree of Photoreactive
Crosslinking Group
[0065] The ratio of a photoreactive group (photoreactive
crosslinking group) introduced into a polysaccharide means a value
expressed by a percentage of the number of photoreactive groups
introduced per repeating disaccharide unit of the polysaccharide.
An amount of the polysaccharide necessary for calculating the
substitution degree was measured by a carbazole assay making use of
a calibration curve, and an amount of the photoreactive residue in
the case of the use of cinnamic acid or amino-cinnamic acid as the
photoreactive residue was measured by an absorbance assay
(measuring wavelength 269 nm) making use of a calibration curve.
However, when the carbazole assay could not be used, this was
measured by a dry loss assay making use of a calibration curve.
MEASUREMENT EXAMPLE 2
Method for Measuring Crosslinking Ratio
[0066] Regarding the crosslinking ratio, 1 g of each substance to
be tested was saponified for 1 hour with 1 ml of 1 mol/l sodium
hydroxide, the thus obtained solution was acidified, and then the
substances derived from the photoreactive group (monomer and dimer
of the photoreactive residue) were extracted with ethyl acetate and
analyzed by high performance liquid chromatography (HPLC) to
measure an amount of the dimer making use of a calibration curve.
Thereafter, the number of moles of the dimerized photoreactive
group per the photoreactive group introduced into the
polysaccharide was calculated by a percentage (%).
[0067] In the following Examples, "%" means "% by weight" unless
otherwise indicated.
EXAMPLE 1
Composition of the Present Invention which Uses Chondroitin
Sulfate
[0068] (1) In 100 ml of distilled water, 1 g of shark cartilage
chondroitin sulfate (also to be called CS in this description,
trade name: Chondroitin Sulfate C, manufactured by Seikagaku
Corporation, weight average molecular weight: 45,000) was dissolved
and 50 ml of 1,4-dioxane was added thereto. Subsequently, 182 mg of
N-hydroxysuccinimide (hereinafter also referred to as "HOSu"), 152
mg of 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide hydrochloride
(hereinafter also referred to as "EDCI.HCl") and 192 mg of
aminopropyl cinnamate hydrochloride
(HCl.H.sub.2N(CH.sub.2).sub.3OCOCH.dbd.CH-Ph: Ph represents a
phenyl group) were added thereto in this order to carry out the
reaction at room temperature for 2 hours, 1 g of sodium chloride
(hereinafter referred also to as "NaCl") was added thereto, and
then the mixture was poured into ethanol to obtain a precipitate.
The precipitate was washed and recovered and then dried at
40.degree. C. under a reduced pressure to obtain 1 g of
photoreactive CS in which HN(CH).sub.3OCOCH.dbd.CH--PH was bound to
the carboxyl group of CS. The substitution degree of the
photoreactive group into the photoreactive CS, measured in
accordance with Measurement Example 1, was 5.3%. (2) The
photoreactive CS obtained in the above-described (1) was dissolved
in 5 mmol/l phosphate buffer to give a weight concentration of 3%,
and then d-sorbitol was dissolved therein to give a weight
concentration of 6% to thereby obtain a reaction solution. The
reaction solution was filtered through a 0.22 .mu.m membrane filter
(manufactured by Nippon Millipore) and then poured onto a glass
plate which had been adjusted to a void of 1 mm, and the reaction
solution was frozen under an atmosphere of -20.degree. C. While
keeping the frozen state, ultraviolet ray irradiation was carried
out at an irradiation light dose of 5000 mJ/cm.sup.2 using an 800 W
high pressure mercury lamp (manufactured by Oak Seisakusho). After
the irradiation, the mixture was melted by returning to room
temperature to obtain a gel-state photocrosslinked-CS composition.
Its crosslinking ratio was 33% when measured in accordance with
Measurement Example 2.
[0069] When measurement was carried out by continuously changing
from 0.01 to 10 Hz at 37.degree. C. using a control stress type
rheometer (manufactured by Carri-Med, England: acrylic parallel
plate diameter 4 cm, 0.degree.), the storage modulus of elasticity
(G') of 1 ml sample was from 8.1 to 70.5 Pa, and the loss modulus
of elasticity (G'') was from 5.6 to 29.7 Pa.
[0070] On the other hand, as a control, a solution prepared by
dissolving the photoreactive CS in 5 mmol/l phosphate buffer to
give a weight concentration of 3% was subjected to
photo-crosslinking reaction in the same manner as in the above, and
then the product was melted by returning to room temperature to
obtain photocrosslinked-CS. The photocrosslinked-CS showed a spongy
state, so that a gel-state photocrosslinked-CS was not
obtained.
EXAMPLE 2
Composition of the Present Invention which Uses Dermatan
Sulfate
[0071] (1) In 100 ml of distilled water, 1 g of cockscomb dermatan
sulfate
[0072] (manufactured by Seikagaku Corporation, weight average
molecular weight: 30,000, hereinafter also referred to as "DS") was
dissolved, 50 ml of 1,4-dioxane was added thereto, 46 mg of HOSu,
38 mg of EDCI.HCl and 48 mg of aminopropyl cinnamate hydrochloride
were added thereto in this order to carry out the reaction at room
temperature for 2 hours, and 1 g of sodium chloride was added
thereto, and then the mixture was poured into ethanol to obtain a
precipitate. The precipitate was washed and recovered and then
dried at 40.degree. C. under a reduced pressure to obtain 1 g of
photoreactive DS in which HN(CH).sub.3OCOCH.dbd.CH--PH was bound to
the carboxyl group. The substitution degree of the photoreactive
group into the photoreactive DS was 5.8% when measured in
accordance with Measurement Example 1.
(2) The photoreactive DS obtained in the above-described (1) was
dissolved in 5 mmol/l phosphate buffer to give a weight
concentration of 3%, and then d-sorbitol was dissolved therein to
give a weight concentration of 1.5% to thereby obtain a reaction
solution. The reaction solution was subjected to photo-crosslinking
reaction at an irradiation light dose of 7000 mJ/cm.sup.2 in the
same manner as in Example 1, and after the irradiation, the mixture
was melted by returning to room temperature to obtain a gel-state
photocrosslinked-DS composition. Its crosslinking ratio was 36%
when measured in accordance with Measurement Example 2.
[0073] On the other hand, as a control, a solution prepared by
dissolving the photoreactive DS in 5 mM phosphate buffer to give a
weight concentration of 3% was subjected to photo-crosslinking
reaction in the same manner as in the above, and then the product
was melted to obtain a photocrosslinked-DS, but the thus obtained
photocrosslinked-DS showed a spongy state, so that gel-state
photocrosslinked-DS was not obtained.
EXAMPLE 3
Composition of the Present Invention which Uses a Heparin
Derivative
[0074] (1) In 100 ml of distilled water, 3 g of a derivative of
swine small intestine heparin, 6-O-desulfated heparin (a heparin
derivative prepared in accordance with the method described in
WO00/06608, weight average molecular weight: about 9,000,
hereinafter also referred to as "6-O-desulfated Hep") was
dissolved, 50 ml of 1,4-dioxane was added thereto, 615.8 mg of
HOSu, 513.4 mg of EDCI.HCl and 648.1 mg of aminopropyl cinnamate
hydrochloride were added thereto in this order to carry out the
reaction at room temperature for 2 hours, and 3 g of NaCl was added
thereto, and then the mixture was poured into ethanol to obtain a
precipitate. The precipitate was washed and recovered and then
dried at 40.degree. C. under a reduced pressure to obtain 2.7 g of
photoreactive 6-O-desulfated Hep. The substitution degree of the
photoreactive group was 65.5% when measured in accordance with
Measurement Example 1. (2) The photoreactive 6-O-desulfated Hep
obtained in the above-described (1) was dissolved in water for
injection to give a weight concentration of 4%, and then d-sorbitol
was dissolved therein to give a weight concentration of 7% to
thereby obtain a reaction solution. The reaction solution was
filtered through a 0.22 .mu.m membrane filter (manufactured by
Nippon Millipore), poured onto a Pyrex glass plate which had been
adjusted to a void of 1 mm, and then frozen under an atmosphere of
-20.degree. C. While keeping the frozen state, ultraviolet ray was
irradiated at an irradiation light dose of 5000 mJ/cm.sup.2 using
an 800 W high pressure mercury lamp. After the irradiation, the
mixture was melted by returning to room temperature to obtain a
gel-state photocrosslinked-6-O-desulfated Hep composition. The
crosslinking ratio was 67.6% when measured in accordance with
Measurement Example 2. In addition, when the viscosity was measured
using a rotational viscometer (manufactured by TOKIMEC) under
conditions of standard cone (1 degree) and 20.degree. C., viscosity
of 1 ml of the photocrosslinked-6-O-desulfated Hep composition was
9840 mPas.
[0075] On the other hand, as a control, a solution prepared by
dissolving the photoreactive 6-O-desulfated Hep in water for
injection to give a weight concentration of 3% was subjected to
photo-crosslinking reaction in the same manner as in the above and
to the light irradiation, and then the product was melted, but the
thus obtained photocrosslinked-6-O-desulfated Hep showed a spongy
state, so that a gel-state photocrosslinked-6-O-desulfated Hep was
not obtained.
EXAMPLE 4
[0076] (1) In 100 ml of distilled water, 3 g of 6-O-desulfated Hep
was dissolved, 50 ml of 1,4-dioxane was added thereto, 205.8 mg of
HOSu, 171.4 mg of EDCI.HCl and 216.1 mg of aminopropyl cinnamate
hydrochloride were added thereto in this order to carry out the
reaction at room temperature for 2 hours, and 3 g of NaCl was added
thereto, and then the mixture was poured into ethanol to obtain a
precipitate. The precipitate was washed and recovered and then
dried at 40.degree. C. under a reduced pressure to obtain 2.7 g of
photoreactive 6-O-desulfated Hep. The substitution degree of the
photoreactive group was 16.0% when measured in accordance with
Measurement Example 1. (2) The photoreactive 6-O-desulfated Hep
obtained in the above-described (1) was dissolved in water for
injection to give a weight concentration of 4%, and then d-sorbitol
was dissolved therein to give a weight concentration of 7%, or
poly(ethylene glycol) 400 (hereinafter also referred to as "PEG
400") to give a weight concentration of 4% to thereby obtain a
reaction solution. Each of the reaction solutions was subjected to
photo-crosslinking reaction in the same manner as in Example 3(2),
and then the product was melted by returning to room temperature to
obtain a photocrosslinked-6-O-desulfated Hep composition. Both of
the reaction solutions resulted in gel-state
photocrosslinked-6-O-desulfated Hep compositions. Thereafter, the
crosslinking ratio of each photocrosslinked-6-O-desulfated Hep
composition was measured in accordance with Measurement Example 2,
and the viscosity was measured under conditions of standard cone (1
degree) and 20.degree. C. Also, RE-80L was used for the sample
which is obviously liquid, and RE-80U for the gel-state sample. The
results are shown in Table 1. On the other hand, regarding the
reaction solution to which d-sorbitol was added, the viscosity was
measured also on the un-irradiated reaction solution.
TABLE-US-00001 TABLE 1 Viscosity of 1 ml photocross- Viscosity of
linked-6-O- Cross- 1 ml unirradi- desulfated Hep linking ated
reaction Added composition ratio solution Prop- substance (mPa s)
(%) (mPa s) erty d-Sorbitol (7%) 4210.0 45.5 298.0 gel PEG 400 (4%)
3700.0 13.4 not measured gel
[0077] On the other hand, as a control, the photoreactive
6-O-desulfated Hep was dissolved in water for injection to give a
weight concentration of 3% and subjected to photo-crosslinking
reaction in the same manner as in the above. The thus obtained
photocrosslinked-6-O-desulfated Hep showed a spongy state, so that
a gel-state photocrosslinked-6-O-desulfated Hep was not
obtained.
EXAMPLE 5
[0078] (1) In 100 ml of distilled water, 1 g of 6-O-desulfated Hep
was dissolved, 50 ml of 1,4-dioxane was added thereto, 1371 mg of
HOSu, 1142 mg of EDCI.HCl and 1440 mg of aminopropyl cinnamate
hydrochloride were added thereto in this order to carry out the
reaction at room temperature for 3 hours, and 1 g of NaCl was added
thereto, and then the mixture was poured into ethanol to obtain a
precipitate. The precipitate was washed and recovered and then
dried at 40.degree. C. under a reduced pressure to obtain 1.1 g of
photoreactive 6-O-desulfated Hep. The substitution degree was 16.5%
when measured in accordance with Measurement Example 1. (2) The
photoreactive 6-O-desulfated Hep obtained in the above-described
(1) was dissolved in water for injection to give a weight
concentration of 4%, and then PEG 400 was dissolved therein to give
a weight concentration of 4%, and PEG 4000 to give a weight
concentration of 2%, sodium lauryl sulfate (SDS) to give a weight
concentration of 4%, ethylenediaminetetraacetic acid (EDTA) to give
a weight concentration of 4%, or citric acid to give a weight
concentration of 4% or 2% to thereby obtain a reaction solution.
Each of the 6 reaction solutions was subjected to
photo-crosslinking reaction in the same manner as in Example 3(2)
to obtain a photocrosslinked-6-O-desulfated Hep composition.
Thereafter, the viscosity of each of the thus obtained
photocrosslinked-6-O-desulfated Hep compositions was measured using
a rotational viscometer under conditions of standard cone (1
degree) and 20.degree. C. The results are shown in Table 2.
[0079] Also, the viscosity of a solution prepared by dissolving the
photoreactive CS obtained in the above in water for injection to
give a weight concentration of 4% measured in the same manner as in
the above was 76.8 mPas.
TABLE-US-00002 TABLE 2 Added substance Viscosity (mPa s) Property
PEG 400 (4%) 192.0 viscous solution PEG 4000 (2%) 819.2 gel-state
SDS (4%) 1888.0 gel-state EDTA (4%) 6822.0 sponge-state Citric acid
(2%) 7021.0 sponge-state Citric acid (4%) 409.6 gel-state Control
solution 76.8 solution
EXAMPLE 6
[0080] (1) In 150 ml of distilled water, 5 g of shark cartilage CS
(lot number: N-251, manufactured by Seikagaku Corporation, weight
average molecular weight: about 30,000) was dissolved, 75 ml of
1,4-dioxane was added thereto, 13.7 g of HOSu, 11.4 g of EDCI.HCl
and 14.4 g of aminopropyl cinnamate hydrochloride were added
thereto in this order to carry out the reaction at room temperature
for 3 hours, and 4 g of NaCl was added thereto, and then the
mixture was poured into ethanol to obtain a precipitate. The
precipitate was washed and recovered and then dried at 40.degree.
C. under a reduced pressure to obtain 4 g of photoreactive CS. The
substitution degree of the photoreactive group was 32.4% when
measured in accordance with Measurement Example 1. (2) The
photoreactive CS obtained in the above-described (1) was dissolved
in water for injection to give a weight concentration of 4%, PEG
400, PEG 4000 or citric acid was added thereto to give a weight
concentration of 2% or 4%, or SDS or EDTA was added thereto to give
a weight concentration of 4%, and then d-sorbitol was added thereto
to give a weight concentration of 8% to thereby obtain a reaction
solution. Each of these 9 reaction solutions was subjected to
photo-crosslinking reaction in the same manner as in Example 3(2)
to obtain a photocrosslinked-CS composition. Thereafter, the
crosslinking ratio was measured in accordance with Measurement
Example 2, and the viscosity was measured using a rotational
viscometer under conditions of standard cone (1 degree) and
20.degree. C. The viscosity was measured also on the reaction
solutions before the crosslinking reaction. The results are shown
in Table 3.
[0081] On the other hand, as a control, the photoreactive CS was
dissolved in water for injection to give a weight concentration of
4% and subjected to photo-crosslinking reaction in the same manner
as in the above. The thus obtained photocrosslinked-CS showed a
spongy state, so that gel-state photocrosslinked-CS was not
obtained.
TABLE-US-00003 TABLE 3 Viscosity of Viscosity of photocross-
Crosslinking unirradiated linked-CS ratio of photo- reaction
composition crosslinked-CS solution Prop- (mPa s) composition (%)
(mPa s) erty PEG 400 3840.0 13.27 39.0 gel (2%) PEG 400 222.6 11.08
27.6 gel (4%) PEG 4000 7117.0 16.02 37.2 gel (2%) PEG 4000 99.6
7.47 31.8 gel (4%) Citric 16540.0 25.55 34.8 gel acid (2%) Citric
7117.0 22.29 28.8 gel acid (4%) SDS (4%) 27290.0 17.57 39.0 sponge
EDTA 19660.0 37.03 28.8 gel (4%) d-Sorbitol 28980.0 29.85 33.0 gel
(8%) Control 38140.0 32.90 not measured sponge
[0082] Appearances of the photocrosslinked-CS compositions obtained
by adding each of PEG 400 (2%), PEG 4000 (2%), citric acid (2%),
citric acid (4%), SDS (4%), EDTA (4%) and d-sorbitol (8%), and the
control photocrosslinked-CS composition are shown in FIG. 1
(photograph).
[0083] In addition, an oscillation test was carried out on the
photocrosslinked-polysaccharide compositions obtained by adding
each of PEG 400 (2%), PEG 4000 (2%) and citric acid (4%), and on
the control. That is, using a control stress regulation type
rheometer (RA 2000, manufactured by TA Instrument), the measurement
was carried out at 37.degree. C. by continuously changing from 10
Hz to 0.01 Hz to measure the storage modulus of elasticity (G') and
the loss modulus of elasticity (G'') of 1 ml samples, and the tan
.delta. (G''/G') generally used as the index of viscoelasticity was
calculated (FIG. 2).
[0084] As a result of the oscillation test, the storage modulus of
elasticity (G') was larger than the loss modulus of elasticity
(G'') (G'>G'') in each case, that is, it shows that the
substances obtained in this example are substances which have high
elasticity (G') and low viscosity (G'').
EXAMPLE 7
[0085] (1) In 150 ml of distilled water, 5 g of swine small
intestine heparin (manufactured by SPF, USA, weight average
molecular weight: about 9,000, hereinafter also referred to as
"Hep") was dissolved, 75 ml of 1,4-dioxane was added thereto, 615
mg of HOSu, 513.4 mg of EDCI.HCl and 648.1 mg of aminopropyl
cinnamate hydrochloride were added thereto in this order to carry
out the reaction at room temperature for 2 hours, and 3 g of NaCl
was added thereto, and then the mixture was poured into ethanol to
obtain a precipitate. The precipitate was washed and recovered and
then dried at 40.degree. C. under a reduced pressure to obtain 4 g
of photoreactive Hep. The substitution degree of the photoreactive
group was 47.4% when measured in accordance with Measurement
Example 1. (2) The photoreactive Hep obtained in the
above-described (1) was dissolved in water for injection to give a
weight concentration of 4%, PEG 400, PEG 4000, citric acid, SDS,
EDTA or d-sorbitol was added thereto to give the respective
concentration described in Table 4 and dissolved to thereby obtain
a reaction solution. Each of these 8 reaction solutions was
subjected to photo-crosslinking reaction in the same manner as in
Example 6 to obtain a photocrosslinked-Hep composition, and the
crosslinking ratio and viscosity were measured. The viscosity was
measured also on the un-irradiated reaction solutions before the
crosslinking reaction. The results are shown in Table 4.
[0086] On the other hand, as a control, the photoreactive Hep was
dissolved in water for injection to give a weight concentration of
4% and subjected to photo-crosslinking reaction in the same manner
as in the above. The thus obtained photocrosslinked-Hep showed a
spongy state, so that gel-state photocrosslinked-Hep was not
obtained.
TABLE-US-00004 TABLE 4 Viscosity of Viscosity of photocross-
Crosslinking unirradiated linked-Hep ratio of photo- reaction
composition crosslinked-Hep solution Prop- (mPas) composition (%)
(mPa s) erty PEG 400 153.6 18.84 10.8 gel (2%) PEG 400 37.2 12.89
14.4 gel (4%) PEG 4000 1587.0 20.08 32.4 gel (2%) PEG 4000 43.8
8.78 24.6 gel (4%) Citric 922.0 20.58 14.4 gel acid (2%) Citric
33.0 15.42 16.2 gel acid (4%) SDS (4%) 6451.0 24.56 11.4 gel EDTA
1946.0 28.53 14.4 gel (4%) d-Sorbitol 39.6 20.61 16.2 gel (8%)
Control 10650.0 31.17 Not measured sponge
[0087] Appearances of the photocrosslinked-CS compositions obtained
by adding each of PEG 4000 (2%), SDS (4%) and EDTA (4%), and the
control photocrosslinked-CS composition are shown in FIG. 3
(photograph).
EXAMPLE 8
[0088] (1) In 150 ml of distilled water, 1 g of
carboxymethylcellulose (manufactured by Nacalai Tesque, hereinafter
also referred to as "CMC") was dissolved, 75 ml of 1,4-dioxane was
added thereto, 324 mg of HOSu, 285 mg of EDCI.HCl and 350 mg of
aminopropyl cinnamate hydrochloride were added thereto in this
order to carry out the reaction at room temperature for 3 hours,
and 1 g of NaCl was added thereto, and then the mixture was poured
into 600 ml of ethanol to obtain a precipitate. The precipitate was
washed and recovered and then dried at 40.degree. C. under a
reduced pressure to obtain 1 g of photoreactive CMC. The
substitution degree of the photoreactive group was 19.5%. (2) The
photoreactive CMC obtained in the above-described (1) was dissolved
in water for injection to give a weight concentration of 4%, and
then PEG 400 was dissolved therein to give a weight concentration
of 4%, or d-sorbitol to give a weight concentration of 8% to
thereby obtain a reaction solution. When each of these reaction
solutions was subjected to photo-crosslinking reaction in the same
manner as in Example 3(2), a gel-state photocrosslinked-CMC
composition was obtained in each case.
[0089] On the other hand, when the photoreactive CMC was dissolved
in water for injection to give a weight concentration of 4% and
subjected as a control to the photo-crosslinking reaction in the
same manner as in the above, a spongy photocrosslinked-CMC was
obtained.
EXAMPLE 9
[0090] Each of the two gel-like photocrosslinked-CMC compositions
obtained in Example 8 and the spongy photocrosslinked-CMC was
filled in a 1 ml capacity syringe in an amount of about 0.3 ml
(about 30 mm), and the mixture was used as the substance to be
tested. Then, 0.2 ml of Blue Dextran solution was gently added from
the upper side to each substance to be tested, and then
centrifugation of 4000 rpm.times.3 minutes was carried out. Also, 1
ml of the 1 ml capacity syringe can be converted to 1 cm, so that
one scale is 1 mm. The permeation ratio is expressed by the scale
width from the contacting surface with the Blue Dextran solution.
As a result, Blue Dextran was permeated into 25 mm in the case of
the spongy photocrosslinked-CMC, but permeation of Blue Dextran was
inhibited in the case of the gel-state photocrosslinked-CMC
composition, which was 15 mm in the case of the
photocrosslinked-CMC composition obtained by adding PEG 400 (4%),
or 4 mm in the case of the photocrosslinked-CMC composition
obtained by adding d-sorbitol (8%).
[0091] In addition, each of the gel-state photocrosslinked-CMC
compositions obtained in Example 6 by adding each of d-sorbitol
(8%) and PEG 4000 (2%), and the spongy photocrosslinked-CMC used as
the control was filled in a 1 ml capacity syringe in an amount of
about 0.4 ml. Using the mixture as the substance to be tested, the
permeation ratio of the Blue Dextran solution was measured in the
same manner as in the above. As a result, it was 5 mm from the
contacting surface with the Blue Dextran solution in the case of
the photocrosslinked-CMC composition obtained by adding d-sorbitol
(8%), or 4 mm in the case of the photocrosslinked-CMC composition
obtained by adding PEG 4000 (2%), so that permeation of Blue
Dextran was inhibited in each case. However, the value was 17 mm in
the case of the spongy photocrosslinked-CMC, showing high
permeability of Blue Dextran.
[0092] While the invention has been describe in detail and with
reference to specific embodiments thereof, it will be apparent to
one skilled in the art that various changes and modifications can
be made therein without departing from the spirit and scope of the
present invention.
[0093] This application is based on a Japanese patent application
filed on Mar. 11, 2003 (Japanese Patent Application No.
2003-065704), the contents thereof being thereby incorporated by
reference. All of the references cited therein are incorporated as
a whole.
INDUSTRIAL APPLICABILITY
[0094] A novel photocrosslinked-polysaccharide composition and
process for producing the same are provided by the present
invention. According to the production process, a photoreactive
polysaccharide can be efficiently photo-crosslinked. In addition,
according to the production process, a gel-state
photocrosslinked-polysaccharide composition capable of being
injected by an injector can also be obtained easily, so that it can
be used in medical applications and the like by using the specific
properties of the gel, and is useful.
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